Early Postnatal Loss Research Articles

Neonatal loss of FGFR2 in astroglial cells affects locomotion, sociability, working memory, and glia-neuron interactions in mice

Fibroblast growth factor receptor 2 (FGFR2) is almost exclusively expressed in glial cells in postnatal mouse brain, but its impact in glia for brain behavioral functioning is poorly understood. We compared behavioral effects from FGFR2 loss in both neurons and astroglial cells and from FGFR2 loss in astroglial cells by using either the pluripotent progenitor-driven hGFAP-cre or the tamoxifen-inducible astrocyte-driven GFAP-creERT2 in Fgfr2 floxed mice. When FGFR2 was eliminated in embryonic pluripotent precursors or in early postnatal astroglia, mice were hyperactive, and had small changes in working memory, sociability, and anxiety-like behavior. In contrast, FGFR2 loss in astrocytes starting at 8 weeks of age resulted only in reduced anxiety-like behavior. Therefore, early postnatal loss of FGFR2 in astroglia is critical for broad behavioral dysregulation. Neurobiological assessments demonstrated that astrocyte-neuron membrane contact was reduced and glial glutamine synthetase expression increased only by early postnatal FGFR2 loss. We conclude that altered astroglial cell function dependent on FGFR2 in the early postnatal period may result in impaired synaptic development and behavioral regulation, modeling childhood behavioral deficits like attention deficit hyperactivity disorder (ADHD).

Preserved Motility after Neonatal Dopaminergic Lesion Relates to Hyperexcitability of Direct Pathway Medium Spiny Neurons.

In Parkinson's disease patients and rodent models, dopaminergic neuron loss (DAN) results in severe motor disabilities. In contrast, general motility is preserved after early postnatal DAN loss in rodents. Here we used mice of both sexes to show that the preserved motility observed after early DAN loss depends on functional changes taking place in medium spiny neurons (MSN) of the dorsomedial striatum (DMS) that belong to the direct pathway (dMSN). Previous animal model studies showed that adult loss of dopaminergic input depresses dMSN response to cortical input, which likely contributes to Parkinson's disease motor impairments. However, the response of DMS-dMSN to their preferred medial PFC input is preserved after neonatal DAN loss as shown by in vivo studies. Moreover, their response to inputs from adjacent cortical areas is increased, resulting in reduced cortical inputs selectivity. Additional ex vivo studies show that membrane excitability increases in dMSN. Furthermore, chemogenetic inhibition of DMS-dMSN has a more marked inhibitory effect on general motility in lesioned mice than in their control littermates, indicating that expression of normal levels of locomotion and general motility depend on dMSN activity after early DAN loss. Contrastingly, DMS-dMSN inhibition did not ameliorate a characteristic phenotype of the DAN-lesioned animals in a marble burying task demanding higher behavioral control. Thus, increased dMSN excitability likely promoting changes in corticostriatal functional connectivity may contribute to the distinctive behavioral phenotype emerging after developmental DAN loss, with implications for our understanding of the age-dependent effects of forebrain dopamine depletion and neurodevelopment disorders.SIGNIFICANCE STATEMENT The loss of striatal dopamine in the adult brain leads to life-threatening motor impairments. However, general motility remains largely unaffected after its early postnatal loss. Here, we show that the high responsiveness to cortical input of striatal neurons belonging to the direct basal ganglia pathway, crucial for proper motor functioning, is preserved after early dopamine neuron loss, in parallel with an increase in these cells' membrane excitability. Chemogenetic inhibition studies show that the preserved motility depends on this direct pathway hyperexcitability/hyperconnectivity, while other phenotypes characteristic of this condition remained unaltered despite the dMSN inhibition. This insight has implications for our understanding of the mechanism underlying the behavioral impairments observed in neuropsychiatric conditions linked to early dopaminergic hypofunction.

Postnatal Sox6 Regulates Synaptic Function of Cortical Parvalbumin-Expressing Neurons.

Cortical parvalbumin-expressing (Pvalb+) neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we used conditional knock-out strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphologic synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.SIGNIFICANCE STATEMENT Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here, we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.

Abstract 5096: Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN

Abstract Renal cell carcinoma (RCC) occurs in up to 4% of tuberous sclerosis complex (TSC) patients, often affecting children and young adults. The mechanisms of TSC-associated RCC are not well understood. We explored the pathogenesis of TSC-associated RCC using three mouse models. First, loss of Tsc2 was induced by i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 (E17.5) in Tsc2flox/floxROSA26-CreER+ mice (n=24), examined at age 6 m. Second, loss of Tsc1 was induced by injecting Cre-encoding adenovirus directly into the left kidney of Tsc1flox/flox mice at P1 (n=19), examined at age 1 year. Third, Tsc2 was knock out through 5 consecutive times of i.p. tamoxifen injection (1mg/mouse/day) when the Tsc2flox/floxROSA26-CreER+ mice were 6 weeks old (n=7), examined at age 6 month. We found the following: In the E17.5 TM injection mouse model, each kidney had more than 200 tumor lesions, which almost occupied the whole kidney cortex and medulla. Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney. The same types of tumor but fewer tumor lesions (~40/kidney) were found in the Cre-encoding adenovirus group. All the females in the P6 week group had the same phenotype as E17.5 TM injection group, but only single cyst was found in the male kidneys. So, the earlier those mice lost TSC1/2, the more severe their tumor burdens. IHC was performed to further validate the diagnosis. All the PRCC showed strong and diffuse expression of phospho-S6-S240/244 (pS6), CK7, and carbonic anhydrase-IX, with negative vimentin staining, while all the HOCT were positive for pS6, S-100 (a diagnostic marker used for human HOCT) and vimentin. The single cysts in the P6 week TM injection male kidneys only showed pS6 and CK7 staining. To further explore the mechanism of tumorigenesis, a phospho-RTK screen was done using lysates from kidney tumors (PRCC and HOCT) and normal kidneys. The pAXL level was 2.8-fold higher in comparison to normal kidney. Immunoblotting revealed high levels of p-AXL and total AXL in kidney tumor lysates compared to normal kidney. AXL mRNA, measured by real-time RT-PCR, was increased 2-fold (p=0.011). c-Jun, a known driver of AXL expression, was also highly expressed in those tumors but negative in normal kidneys as shown by immunoblot. Immunoblot analysis of a cell line established from the kidney tumors showed TSC-dependent c-Jun and AXL expression. These novel mouse models of TSC-related RCC closely resemble those found in TSC patients and elucidate distinct mechanisms of disease development and potential therapeutic targets. Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5096.

Conditional Disabled-1 Deletion in Mice Alters Hippocampal Neurogenesis and Reduces Seizure Threshold.

Many animal models of temporal lobe epilepsy (TLE) exhibit altered neurogenesis arising from progenitors within the dentate gyrus subgranular zone (SGZ). Aberrant integration of new neurons into the existing circuit is thought to contribute to epileptogenesis. In particular, adult-born neurons that exhibit ectopic migration and hilar basal dendrites (HBDs) are suggested to be pro-epileptogenic. Loss of reelin signaling may contribute to these morphological changes in patients with epilepsy. We previously demonstrated that conditional deletion of the reelin adaptor protein, disabled-1 (Dab1), from postnatal mouse SGZ progenitors generated dentate granule cells (DGCs) with abnormal dendritic development and ectopic placement. To determine whether the early postnatal loss of reelin signaling is epileptogenic, we conditionally deleted Dab1 in neural progenitors and their progeny on postnatal days 7–8 and performed chronic video-EEG recordings 8–10 weeks later. Dab1-deficient mice did not have spontaneous seizures but exhibited interictal epileptiform abnormalities and a significantly reduced latency to pilocarpine-induced status epilepticus. After chemoconvulsant treatment, over 90% of mice deficient for Dab1 developed generalized motor convulsions with tonic-clonic movements, rearing, and falling compared to <20% of wild-type mice. Recombination efficiency, measured by Cre reporter expression, inversely correlated with time to the first sustained seizure. These pro-epileptogenic changes were associated with decreased neurogenesis and increased numbers of hilar ectopic DGCs. Interestingly, neurons co-expressing the Cre reporter comprised a fraction of these hilar ectopic DGCs cells, suggesting a non-cell autonomous effect for the loss of reelin signaling. We also noted a dispersion of the CA1 pyramidal layer, likely due to hypomorphic effects of the conditional Dab1 allele, but this abnormality did not correlate with seizure susceptibility. These findings suggest that the misplacement or reduction of postnatally-generated DGCs contributes to aberrant circuit development and hyperexcitability, but aberrant neurogenesis after conditional Dab1 deletion alone is not sufficient to produce spontaneous seizures.

Strain-Specific Defects in Testicular Development and Sperm Epigenetic Patterns in 5,10-Methylenetetrahydrofolate Reductase-Deficient Mice

Methylenetetrahydrofolate reductase (MTHFR) is a crucial folate pathway enzyme that contributes to the maintenance of cellular pools of S-adenosylmethionine, the universal methyl donor for several reactions including DNA methylation. Whereas Mthfr(-/-) BALB/c mice show growth retardation, developmental delay, and spermatogenic defects and infertility, C57BL/6 mice appear to have a less severe phenotype. In the present study, we investigated the effects of MTHFR deficiency on early germ cell development in both strains and assessed whether MTHFR deficiency results in DNA methylation abnormalities in sperm. The reproductive phenotype associated with MTHFR deficiency differed strikingly between the two strains, with BALB/c mice showing an early postnatal loss of germ cell number and proliferation that was not evident in the C57BL/6 mice. As a result, the BALB/c MTHFR-deficient mice were infertile, whereas the C57BL/6 mice had decreased sperm numbers and altered testicular histology but showed normal fertility. Imprinted genes and sequences that normally become methylated during spermatogenesis were unaffected by MTHFR deficiency in C57BL/6 mice. In contrast, a genome-wide restriction landmark genomic scanning approach revealed a number of sites of hypo- and hypermethylation in the sperm of this mouse strain. These results showing strain-specific defects in MTHFR-deficient mice may help to explain population differences in infertility among men with common MTHFR polymorphisms.

Early Postnatal Loss of Heat Sensitivity Among Cutaneous Myelinated Nociceptors in Swiss–Webster Mice

Cutaneous myelinated nociceptors are known to exhibit considerable heterogeneity in their response to noxious heat. In the present experiments, we studied heat sensitivity among myelinated nociceptors during early postnatal life to determine whether this heterogeneity is correlated with other physiological and anatomical properties. A total of 129 cutaneous myelinated nociceptors were recorded intracellularly and characterized using mechanical and thermal skin stimuli in ex vivo preparations from neonatal Swiss-Webster (SW) mice across postnatal ages P2-P10; physiologically identified cells were iontophoretically labeled with neurobiotin for analyses of dorsal horn terminations from heat-sensitive and heat-insensitive cells. Our results show that heat sensitivity is not strictly correlated with other physiological or anatomical properties, most notably mechanical threshold or laminar termination patterns, of myelinated nociceptors at these ages. Further, we found a marked decline in the number of heat-sensitive myelinated mechanonociceptors (A-mechanoheat nociceptors [AMHs]) during this early postnatal period. Indeed, 68% of myelinated nociceptors were AMHs between P2 and P5, whereas this percentage dropped to 36% between P6 and P10. Multiple independent lines of evidence suggest that this decrease reflects a change in phenotype in a subset of myelinated nociceptors that lose sensitivity to noxious heat in early postnatal life. Interestingly, evidence was also obtained for a significant strain difference since the early transient excess in the number of AMHs in P2-P5 SW neonates was not present in similarly aged neonates from the C57Bl/6 strain. Potential mechanisms underlying these postnatal changes in AMH number are discussed.

Muscle ultrasound density in human fetuses with spina bifida aperta

Background In fetal spina bifida aperta (SBA), leg movements caudal to the meningomyelocele (MMC) are transiently present, but they disappear shortly after birth. Insight in the underlying mechanism could help to improve treatment strategies. In fetal SBA, the pathogenesis of neuromuscular damage prior to movement loss is still unknown. We reasoned that prenatal assessment of muscle ultrasound density (fetal-MUD) could help to reveal whether progressive neuromuscular damage is present in fetal SBA, or not. Aim To reveal whether prenatal neuromuscular damage is progressively present in SBA. Patients/methods In SBA fetuses ( n = 6; 22–37 weeks gestational age), we assessed fetal-MUD in myotomes caudal to the MMC and compared measurements between myotomes cranial to the MMC and controls ( n = 11; 17–36 weeks gestational age). Furthermore, we intra-individually compared MUD and muscle histology between the pre- and postnatal period. Results Despite persistently present fetal leg movements caudal to the MMC, fetal-MUD was higher caudal to the MMC than in controls ( p < 0.05). Fetal-MUD caudal to the MMC did not increase with gestational age, whereas fetal-MUD in controls and cranial to the MMC increased with gestational age ( p < 0.05). In 5 of 6 patients assessed, comparison between pre- and postnatal MUD and/or muscle histology indicated consistent findings. Conclusions In fetal SBA, persistent leg movements concur with stable, non-progressively increased fetal-MUD. These data may implicate that early postnatal loss of leg movements is associated with the impact of additional neuromuscular damage after the prenatal period.

Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.